Our laboratory is studying the 70-kDa class of heat shock proteins (Hsp70s) which act as molecular chaperones, that is, are involved in the ATP-dependent folding and unfolding of proteins, the formation and dissolution of protein complexes, and the translocation of proteins across membranes. In many of these processes members of the DnaJ class of proteins act as necessary cofactors with the Hsc70 proteins, presenting protein substrates to Hsc70. One of the processes where a DnaJ homolog is required for Hsc70 action is the uncoating of clathrin- coated vesicles. In studying the mechanism of uncoating, we discovered that the nerve-specific 100 kDa clathrin assembly protein, auxilin, is required for the uncoating process to occur, and we also showed that auxilin is a DnaJ homolog. We have now found that GAK, a 150 kDa protein recently cloned from both humans and rats, has a C-terminal domain that shows a high homology to auxilin. In vitro, GAK, like auxilin, supports the uncoating of clathrin baskets by Hsc70. In addition, GAK is present in clathrin-coated vesicles isolated from rat liver that can be uncoated by Hsc70. Therefore, GAK is apparently the DnaJ homolog that supports uncoating of non-neuronal clathrin-coated vesicles by Hsc70. We have also found that C. elegans produces a 107 kDa protein that is homologous to auxilin. Recombinant C. elegans auxilin is able to substitute for mammalian auxilin in its interaction with Hsc70 and clathrin in vitro. Furthermore, when RNA-mediated gene interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes in Ce-auxilin (RNAi) worms have reduced uptake of vitellogenin-GFP from the pseudocoelom and F1 Ce-auxilin (RNAi) larvae have many prominent puncta containing large amounts of clathrin heavy chain-GFP compared to wild type. Moreover, all of the F1 Ce-auxilin (RNAi) worms arrested development at the first larval stage, L1. Therefore C. elegans auxilin is required for clathrin-mediated endocytosis in vivo and the C. elegans auxilin gene is essential for viability. Finally we have been studying the ability of Hsp70s mutated at their active sites to carry out various functions of wild type Hsp70. DnaJ homologs induce wild type Hsc70 to rapidly hydrolyze ATP and it is thought that this is a key step in the ability of DnaJ homologs to present substrates to Hsp70. However, we now find that auxilin can induce Hsp70 mutants that cannot hydrolyze ATP to bind to clathrin baskets showing that the key step in the presentation of substrates to Hsp70 is the ability of the DnaJ homolog to transiently open up the Hsp70 substrate binding site rather than to activate the ATP hydrolysis step. - 70-kDa class of heat shock proteins act as molecular chaperones, involved in the ATP-dependent of proteins.